B cells are a critical branch of the immune system and drive antibody-based protection. The strength of an antibody response is determined by the ability of B cells to extract and internalize membrane-bound antigens from antigen-presenting cells (APCs). Antigen uptake requires the formation of an immune synapse (IS) where B cell receptor-bound antigens are moved centripetally into a central cluster before being extracted from the APC and internalized. The force provided by the actin motor myosin 2A (M2A) powers antigen extraction. B cells that lack M2A activate aberrantly and mount weak antibody responses. However, the organization of the actomyosin network, its role in IS formation, and the mechanism by which M2A powers antigen extraction in B cells are unknown. Here we test the hypothesis that the actomyosin network drives the events of IS formation that promote B cell activation and antigen uptake. We first define the dynamic organization of actin and M2A at the IS using the super-resolution imaging modalities TIRF/SIM, 3D-SIM and Airyscan. On functionalized glass and planar lipid bilayers, the A20 B cell line forms concentric arcs in the medial portion of the IS. These arcs are rich in M2A based on immunostaining, and on imaging cells in which endogenous M2A was tagged with GFP using CRISPR. 3D-SIM imaging of APCs with primary splenic B cells isolated from M2A-GFP knock-in mice shows that M2A polarizes towards the IS. B cell IS studies are often performed using antigen stimulation only. in vivo, B cell integrins provide adhesion to APCs and, via an unknown mechanism, allow for IS formation and antigen uptake with weakly-stimulating antigens. Surprisingly, we found that actomyosin arc formation in primary B cells requires both antigen and integrin costimulation, conditions that reflect physiological B cell activation. These contractile actomyosin arcs are especially prominent in primary B cells such that the actomyosin arcs are the major actin structure at the IS. The contractile nature of the actomyosin arcs that dominate at the primary B cell IS may explain why integrin costimulation boosts B cell responses to weakly-stimulating antigens. Notably, integrin costimulation on bilayers produces actin arcs that sweep peripheral antigen clusters centripetally and is required for contracting low amounts of antigen to form the IS. Moreover, M2A inhibition abrogates the organization of actin arcs and prevents antigen centralization. Therefore, we have identified in primary B cells a novel actomyosin network, which comprises the major actin structure at the IS and promotes robust antigen centralization during IS formation. Current efforts are directed at defining the mechanism by which the actomyosin network drives antigen extraction from APCs using live-cell volumetric imaging. T cells are a critical arm of the adaptive immune system because they kill virally-infected or transformed cells and facilitate the function of other immune cells (Zhang & Bevan, 2011; Zhu et al., 2010). T cell dysfunction can lead to an array of severe pathologies including susceptibility to infection, lymphoproliferative disease, autoimmunity, and hypersensitivity (Walter & Santamaria, 2005; Zhu & Paul, 2008). T cell activation is a complex process involving recognition by the T cells unique T cell receptor (TCR) of specific peptide antigen bound to major histocompatibility complex (MHC) on the surface of an antigen-presenting cell (APC). This recognition can lead to long-term stable engagement with the APC and the formation of a highly-organized structure at the T cell: APC interface termed the immunological synapse (IS) (Dustin & Baldari, 2017). The IS itself is a multidomain structure divided into distal, peripheral, and central supramolecular activation complexes (dSMAC, pSMAC, cSMAC). TCR microclusters contact antigen-bearing MHC at the periphery of the IS and are then transported across the dSMAC and pSMAC to the cSMAC. This centripetal movement of microclusters is driven by the retrograde flow of an Arp2/3-generated, branched actin network in the dSMAC and the contraction of formin-generated, myosin 2-rich, concentric actin arcs in the pSMAC (Hammer et al., 2019; Murugesan et al., 2016; Yi et al., 2012; Ditlev et al., pre-print BioRxiv). Perturbation of either of these actin structures dampens TCR signaling and impairs subsequent T cell activation. The goal of this study is to characterize the contributions made by tropomyosin and myosin 18A to the organization, dynamics and function of the actomyosin arcs populating the pSMAC. Tropomyosins are actin-binding proteins that form head-to-tail polymers along the actin filament. Several tropomyosin isoforms have been shown to associate preferentially with linear, formin-generated filaments, where they serve to promote the recruitment and activation of myosin 2 and thwart cofilin-mediated filament disassembly (Gateva et al., 2017; Tojkander et al., 2011). In preliminary experiments, we find that the low-molecular weight tropomyosin isoform 4.1 associates with the pSMAC arcs, and that the tropomyosin inhibitor TR100 disrupts their organization. Myosin 18A is a myosin 2-like protein that lacks motor activity and contains unique N- and C-terminal extensions harboring both recognizable and uncharacterized protein: protein interaction domains. Importantly, myosin 18A co-assembles with myosin 2 to make mixed filaments (Billington et al., 2015), suggesting that myosin 18A serves to recruit proteins to these mixed filaments or attach them to cellular structures. Preliminary experiments show that the myosin 18A isoform myosin 18A is highly expressed in T cells and that it co-assembles with myosin 2 in the pSMAC arcs. Moreover, knockdown or CRISPR-mediated knockout of myosin 18A alters arc organization and attenuates proximal signaling. Current efforts are directed at further clarifying the roles played by tropomyosin and myosin 18A in arc organization and function, as well as in T cell effector functions. Melanoregulin (Mreg), the product of the dilute suppressor locus, is a small, highly-charged, multiply-palmitoylated protein present on the limiting membrane of melanosomes. Previous studies have implicated Mreg in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these and related organelles by binding to RILP, a Rab7 effector that recruits the dynein motor complex. Here we shed new light on the possible molecular function of Mreg by solving its structure using nuclear magnetic resonance (NMR) spectroscopy. Mreg contains six -helices that form an elongated fishhook-like fold in which positive and negative charges occupy opposite sides of the proteins surface and sandwich a putative, tyrosine-based (Y166) cholesterol recognition sequence (CRAC motif). The absence or significant exchange broadening of 1H-15N crosspeaks for multiple residues within this putative CRAC motif and a proximal tryptophan sidechain resonance argue that this motif has functional importance. Consistently, Mreg containing a function blocking point mutation within its CRAC motif (Y166I) still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Moreover, wild type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, reversing the charge of three closely-spaced acidic residues (D177, E180, and D181) also inhibits Mregs ability to drive these organelles to microtubule minus ends, but only partially. We propose that cholesterol recognition alters Mregs orientation on the membrane in such a way as to allow it to interact with a component(s) involved in dynein recruitment (e.g. RILP).